Background: Hepatitis B Virus (HBV) infection is a worldwide biomedical problem and an improved understanding of the assembly and structure of the virus may help develop new antiviral therapies. The HBV core gene codes for precore protein (pre-C) which is either partially processed to form a secreted non-particulate protein called e-antigen (HBeAg) or fully processed to produce core antigen (HBcAg). HBcAg is a 183-residue protein that encapsidate around a RNA-reverse transcriptase complex (HBV polymerase). HBcAg has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues also produces assembly competent protein. The C-terminal truncated protein (Cpe: residues 1-149) does not contain nucleic acid, can be highly purified and is more suitable for structural studies than the full-length protein. Native HBeAg is also C-terminally truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of pre-C. Although the function of HBeAg is unclear it is an important serological marker. ResultsIn earlier work, the structure of Cpe was determined by cryo-electron microscopy and image analysis to a resolution of about 0.8 nm. This allowed various structural domains to be discerned, especially a four alpha-helical bundle which formed the surface projections of the capsid. These surface projections were shown by labeling with monoclonal antibodies to be the site of the immunodominant epitope, an important serological marker. To localize other regions of functional and structural importance, we have introduced by site-directed mutagenesis reactive cysteine residues at various sites in the protein that can be specially labeled with electron dense reagents. Labeling and localizations have been made using novel high-resolution reagents confirming and extending earlier work. After several years of continuous effort, conditions for the crystallization of Cpe have been worked out. Continued effort is being made to improve the quality of the crystals. Other work includes: study of the HBV capsid assembly, results suggests that interfering with assembly could be an important target for antiviral therapeutics; expression of functional regions of the HBV polymerase has been achieved, and we have continue to study the structure of HBeAg. Significance and future direction:The high- resolution structure determination of the HBV capsid using X-ray diffraction appears possible using crystals prepared using the methods developed. The 0.9 nm model derived from cryo-electron microcopy will play a major role in the structural work by providing a suitable molecular phasing model. Other targets of HBV for structural determinations include the HBV polymerase and HBeAg. These proteins, or functional regions thereof, have been expressed in E.coli and structure determinations initiated. SummaryThe Hepatitis B Virus (HBV) is the major worldwide cause of cancer. Although a vaccine is available, chronic HBV is often acquired in childhood. The HBV nucleocapsid plays an important structural role and metabolic role in the life cycle of the virus. An understanding of the molecular structure of the HBV nucleocapsid would allow targeted drug discovery with the aim of preventing the assembly and formation of the virus. - Hepatitis B Virus, nucleocapsid, protein expression, structure determination